The Study Triatoma In 2007, at the site of the first autochthonous case of transmission of Chagas disease in Louisiana, 49 T. san- sanguisuga Blood guisuga (16 male and 33 female) bugs were collected and identified (6–8). DNA was isolated from the abdomen of Meals and each bug by using the DNeasy Blood and Tissue Kit (QIA- Potential for GEN, Valencia, CA, USA), and presence of T. cruzi was assessed by PCR (9). T. cruzi infection was found in 27 Chagas Disease, (55.1%) bugs; prevalence did not differ significantly be- tween males (50.0%, 8/16) and females (57.6%, 19/33) Louisiana, USA (Fisher exact test; p = 0.76) (online Technical Appendix Table, http://wwwnc.cdc.gov/EID/article/20/12/13-1576- 1 Etienne Waleckx, Julianne Suarez, Techapp1.pdf). Bethany Richards, and Patricia L. Dorn Blood meals were detected by using PCR with univer- To evaluate human risk for Chagas disease, we mo- sal vertebrate primers targeting the 12S ribosomal RNA lecularly identified blood meal sources and prevalence of gene (10). The PCR products were purified and cloned to Trypanosoma cruzi infection among 49 Triatoma sanguisu- enable detection of multiple blood sources in a single bug. ga kissing bugs in Louisiana, USA. Humans accounted for For cloning, the p-GEM-T Easy Vector System (Promega, the second most frequent blood source. Of the bugs that Madison, WI, USA) was used; for the ligation step, the fed on humans, ≈40% were infected with T. cruzi, revealing DNA-to-vector ratio was 3:1. After transformation, up to transmission potential. 8 transformants per bug were randomly selected and se- quenced. Blood meals were detected in 45 (92%) of 49 hagas disease, caused by the parasite Trypanosoma bugs, and 43 (96%) of the 45 detected blood meals were Ccruzi, is mainly transmitted to humans and other mam- successfully cloned (online Technical Appendix Table). mals by blood-sucking insects called triatomines (also Blood meal sources were inferred by using BLAST known as kissing bugs). In the United States, 24 species (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi); >97% of wild mammals have been found to be naturally infected identity was considered a match. From the 43 bugs, 201 with T. cruzi, but only a few (<25) autochthonous cases vertebrate 12S sequences were obtained. In all, 8 verte- of vectorial transmission to humans have been described brate blood-source species were identified. Multiple blood (1,2). This number is probably an underestimate, and there source species were identified in 21 (48.8%) of 43 bugs; is concern that vectorial transmission to humans in the Unit- the maximum number of blood meal sources was 4 (online ed States may increase because of the following factors: Technical Appendix Table), confirming the ability of the 1) loss of sylvan blood sources because of habitat destruc- cloning approach to identify multiple blood meals. The av- tion, forcing the bugs to seek other (possibly human) blood erage number of blood source species detected per bug was sources; 2) climate change that could extend the range of 1.6. The predominant blood source was the American green the vectors northward; and 3) introduction of parasites by tree frog (Hyla cinerea), found in 53.5% of triatomines; the migrants from disease-endemic countries (3–5). Among the second most predominant was the human (Homo sapiens), 11 triatomine species in the United States, the most widely found in 48.8%, followed by the raccoon (Procyon lotor), distributed and the only 2 found in Louisiana are Triatoma found in nearly 30% of triatomines (Figure). Less prevalent lecticularia and T. sanguisuga (5). Bugs of the species T. blood sources included cow (Bos taurus), dog/wolf (Canis sanguisuga are responsible for the first described autoch- lupus), squirrel (Sciurus carolinensis), cat (Felis domes- thonous case of T. cruzi transmission in Louisiana (6), but ticus), and woodrat (Neotoma floridana), each found in little is known about their feeding habits in natural condi- <15% of bugs. tions. To evaluate the risk for Chagas disease (based on hu- In total, 33 different vertebrate 12S haplotypes were man/vector/parasite contact) and determine the feeding be- found at an average of 2.1 per bug (online Technical Ap- havior of the species T. sanguisuga, we molecularly identi- pendix Table, Figure). We found 8 human haplotypes, in- fied the blood meal sources and T. cruzi infection in T. dicating that bugs had fed on at least 8 persons, assuming sanguisuga kissing bugs. that multiple haplotypes did not result from heteroplasmy. More female than male bugs had fed on frogs (Fisher ex- act test, p = 0.005). Neither the average number of blood Author affiliation: Loyola University New Orleans, New Orleans, Louisiana, USA 1Current affiliation: Universidad Autónoma de Yucatán, DOI: http://dx.doi.org/10.3201/eid2012.131576 Mérida, México

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Figure. Vertebrate blood meal sources of Triatoma sanguisuga kissing bugs detected by 12S rDNA assay (10). The numbers of triatomines containing each vertebrate blood source are indicated in parenthesis. The numbers of haplotypes of each vertebrate source and the Trypanosoma cruzi infection prevalence in the triatomines containing this vertebrate blood source are indicated.

sources detected (females 1.7 ± 0.8 vs. males 1.4 ± 0.6, is not surprising because these frogs are abundant in this t-test; p = 0.23) nor the average number of vertebrate hap- region (11). Although amphibians are incompetent T. cruzi lotypes found (females 2.0 ± 0.9 vs. males 2.1 ± 0.9, t-test; hosts, frogs contribute to the epidemiology because as a p = 0.75) differed significantly between bugs of each sex. blood source, they help maintain large populations of bugs Of the 55% of bugs that were infected with T. cruzi, near human dwellings. Further investigation could deter- 61.9% had fed on frogs (incompetent T. cruzi host) and mine whether frogs also control the bug population by eat- 38.1% on humans. We found 3 human haplotypes in T. ing them, as do Neotoma spp. rats, the traditional hosts of cruzi–infected bugs, suggesting, in the absence of hetero- kissing bugs in North America (5). Raccoons serve as syl- plasmy, that at least 3 persons were bitten by an infected vatic T. cruzi reservoirs in the southeastern United States bug (online Technical Appendix Table). Because only 2 and play an epidemiologic role because they are frequently persons lived at the location sampled, some bugs may have found close to humans and, as do woodrats, they link the fed on visiting persons or migrated from nearby houses. No sylvatic cycle of the parasite with a domestic cycle (5). significant association was found between infection and a Dogs also serve as reservoirs and are at high risk for Cha- particular blood source, even after we removed from analy- gas disease; many dogs in the southern United States die sis all insects that had fed on at least 1 frog or had fed on of this disease (1,12,13). In addition to the loss of com- frogs only. panion animals, Chagas disease in animals has an evident economic effect (13). Conclusions Results of our study, as well as those of Stevens et al. Our results indicate that T. sanguisuga kissing bugs (10) and Kjos et al. (14), reject the assertion that kissing pose an epidemiologic threat to humans and animals in bugs in North America prefer blood from wild animals, Louisiana. Human/vector/T. cruzi contact is frequent; 55% which has been one explanation for the low prevalence of of bugs were infected, of which nearly 40% had fed on hu- Chagas disease in the United States. Our study provides mans. evidence of frequent vector/human/T. cruzi contact in In addition to humans, the bugs fed on a wide vari- Louisiana and reveals the potential for transmission in the ety of vertebrates; multiple blood sources were detected in United States. Even if this finding does not apply to all lo- about half of the bugs. These observations support catholic calities (e.g., human blood has rarely been detected in tri- and opportunistic feeding habits for T. sanguisuga, which atomines from Texas [15]), the risk for vectorial transmis- probably feed on any available animal. The high occur- sion of T. cruzi in the United States may increase because rence of the American green tree frog as a blood source of expansion of human settlements into formerly sylvatic

2142 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 12, December 2014 Potential for Chagas Disease, Louisiana, USA areas. Moreover, the low number of Chagas disease cases 5. Zeledon R, Beard CB, Diaz JCP, Leiby DA, Dorn PL, Coura JR. An reported in the United States is probably caused, at least in appraisal of the status of Chagas disease in the United States. Oxford (UK): Elsevier; 2012. part, by a lack of awareness (4,5). Because knowledge of 6. Dorn PL, Perniciaro L, Yabsley MJ, Roellig DM, Balsamo G, the feeding behavior of triatomines is critical for the imple- Diaz J, et al. Autochthonous transmission of Trypanosoma cruzi, mentation of efficient control measures, more studies of the Louisiana. Emerg Infect Dis. 2007;13:605–7. http://dx.doi.org/ blood sources of triatomines in North America are needed. 10.3201/eid1304.061002 7. Lent H, Wygodzinsky P. Revision of the triatominae (Hemiptera, In addition, awareness of Chagas disease and surveillance Reduviidae), and their significance as vectors of Chagas’ disease. of insect vectors and human disease should be improved. American Museum of Natural History. 1979;163:321–4. 8. Cesa K, Caillouet KA, Dorn PL, Wesson DM. High Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) prevalence in Triatoma Acknowledgments sanguisuga (Hemiptera: Redviidae) in southeastern Louisiana. We thank Leon Perniciaro, Gabriela Estrada, Laura Duncan, J Med Entomol. 2011;48:1091–4. http://dx.doi.org/10.1603/ME10195 Paula Mischler, and Dawn Wesson for technical and field assistance. 9. Moser DR, Kirchhoff LV, Donelson JE. Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction. Funding was provided by National Institutes of Health grant J Clin Microbiol. 1989;27:1477–82. 1R15 A1079672–01A1, Supervised Undergraduate Research Ex- 10. Stevens L, Dorn PL, Hobson J, de la Rua NM, Lucero DE, Klotz JH, periences from the Louisiana Board of Regents, National Science et al. Vector blood meals and Chagas disease transmission poten- tial, United States. Emerg Infect Dis. 2012;18:646–9. http://dx.doi. Foundation grant BCS-1216193, and the Mullahy, S.J. Fund for org/10.3201/eid1804.111396 Undergraduate Research from Loyola University New Orleans. 11. Dundee HA, Rossman DA. The amphibians and reptiles of Louisiana. Baton Rouge (LA): Louisiana State University Press; 1989. p. 91–2. Dr Waleckx is a research professor in medical entomology at 12. Kjos SA, Snowden KF, Craig TM, Lewis B, Ronald N, Olson JK. the Laboratory of Parasitology of the “Dr. Hideyo Noguchi” Re- Distribution and characterization of canine Chagas disease in gional Research Center, Autonomous University of Yucatán, in Texas. Vet Parasitol. 2008;152:249–56. http://dx.doi.org/10.1016/ j.vetpar.2007.12.021 Mexico. His research interests are focused on the ecology, popu- 13. Nieto PD, Boughton R, Dorn PL, Steurer F, Raychaudhuri S, lation genetics, and control of tropical disease vectors. Esfandiari J, et al. Comparison of two immunochromatographic assays and the indirect immunofluorescence antibody test for di- agnosis of Trypanosoma cruzi infection in dogs in south central References Louisiana. Vet Parasitol. 2009;165:241–7. http://dx.doi.org/10.1016/ j.vetpar.2009.07.010 1. Bern C, Kjos S. Yabsley MJ, Montgomery SP. Trypanosoma 14. Klotz SA, Schmidt JO, Dorn PL, Ivanyi C, Sullivan KR, Stevens L. cruzi and Chagas’ disease in the United States. Clin Microbiol Rev. Free-roaming kissing bugs, vectors of Chagas disease, feed often on 2011;24:655–81. http://dx.doi.org/10.1128/CMR.00005-11 humans in the Southwest. Am J Med. 2014;127:421–6. http://dx.doi. 2. Cantey PT, Stramer SL, Townsend RL, Kamel H, Ofafa K, org/10.1016/j.amjmed.2013.12.017 Todd CW, et al. The United States Trypanosoma cruzi Infec- 15. Kjos SA, Marcet PL, Yabsley MJ, Kitron U, Snowden KF, tion Study: evidence for vector-borne transmission of the parasite Logan KS, et al. Identification of blood meal sources and Trypano- that causes Chagas disease among United States blood donors. soma cruzi infection in triatomine bugs (Hemiptera: Reduviidae) Transfusion. 2012;52:1922–30. http://dx.doi.org/10.1111/j.1537- from residential settings in Texas, the United States. J Med Entomol. 2995.2012.03581.x 2013;50:1126–39. http://dx.doi.org/10.1603/ME12242 3. Waleckx E, Depickere S, Salas R, Aliaga C, Monje M, Calle H, et al. New discoveries of sylvatic Triatoma infestans (Hemiptera: Reduviidae) throughout the Bolivian Chaco. Am J Trop Med Hyg. Address for correspondence: Etienne Waleckx, Centro de Investigaciones 2012;86:455–8. http://dx.doi.org/10.4269/ajtmh.2012.11-0205 Regionales Hideyo Noguchi, Universidad Autónoma de Yucatán, Calle 96 4. Click Lambert R, Kolivras KN, Resler LM, Brewster CC, Paulson SL. The potential for emergence of Chagas disease in the x Av. Jacinto Canek y Calle 47, Col. Paseo de Las Fuentes, C.P. 97225, United States. Geospat Health. 2008;2:227–39. Mérida, Yucatán, México; email: [email protected] Emerging Infectious Diseases Journal Podcasts Think Fungus Dr. Mary Brandt, a CDC research microbiologist, discusses the impact of fungal infections. http://www2c.cdc.gov/podcasts/player.asp?f=8629964

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